title: An expression system of rat calmodulin using T7 phage promoter in Escherichia coli.
abstract: An efficient expression system of rat calmodulin in Escherichia coli is presented. To express rat calmodulin cDNA, we employed a pET expression vector which contains the T7 phage promoter and terminator. After transformation of E. coli BL21(DE3) strain which carries T7 phage RNA polymerase inducible with isopropyl-beta-D-thiogalactopyranoside, induction of the expression, and chromatography of soluble proteins on a phenyl-Sepharose column, about 250 mg of recombinant rat calmodulin was obtained from 1 liter of E. coli culture. The recombinant calmodulin lacked the N-terminal methionine, and posttranslational modifications such as Nalpha-acetylation and methylation. This system facilitates the large amount preparation of calmodulin and the mutant proteins required for the structural analysis by NMR spectrometry and/or X-ray crystallography.
authors.affiliation: Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, 470-11, Japan. nhayashi@fujita-hu.ac.jp
authors.forename: N
authors.initials: N
authors.lastname: Hayashi
authors.forename: M
authors.initials: M
authors.lastname: Matsubara
authors.forename: A
authors.initials: A
authors.lastname: Takasaki
authors.forename: K
authors.initials: K
authors.lastname: Titani
authors.forename: H
authors.initials: H
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D009682:Magnetic Resonance Spectroscopy; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D002147:Calmodulin; D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D009219:Myosin-Light-Chain Kinase